In addition to its role in neurotransmission, 5HT appears to regulate fundamental developmental processes that influence the assembly of brain circuitry. In humans, SERT and 5HT receptor polymorphisms are associated with mental health-related neurodevelopmental disorders, such as autism. In animal models, early 5HT actions include subtle effects on neuronal differentiation, survival and neurite outgrowth, and pronounced effects on sensory map formation. Genetic studies show that disruption of 5HT receptors during a restricted period of development results in long-term behavioral dysfunction. The molecular mechanisms that underlie the 5HT impact on neurodevelopment are unknown, owing to the large number of serotonin receptors and limited understanding of their expression patterns and signal-transducing capabilities. In preliminary studies, Levitt's group has identified a novel role for 5HT in modulating the signaling of classic axon guidance molecules. In Project 2: Serotonin Modulation of Axon Guidance During Brain Development, Pat Levitt and colleagues propose three aims to take advantage of Conte Center collaborations and investigate the role of 5HT in the assembly of two key circuits involved in mental health disorders, thalamocortical axons (TCAs) and dorsal raphe (DR) axons. In Aim I, they propose use of an explant assay system and a novel in utero gene electroporation strategy to establish the mechanisms through which specific 5HT receptor subtypes modulate TCA guidance by Netrin-1. They will identify the impact of 5-HT modulation on the membrane expression of axon guidance receptors that mediate Netrin-1 attraction and repulsion and the role of cyclic nucleotides in mediating modulation. The long term impact of disrupting TCA topography in utero by 5HT receptor knockdown by siRNA or over-expression also will be examined. Detailed analysis of TCA topography will be done in the Pet-1 null mice, in collaboration with Project 1. In Aim II, the mechanisms that govern the guidance of 5-HT-containing DR axons will be studied. In situ hybridization studies will determine the expression patterns of guidance receptors by DR neurons and guidance molecules along their pathway (with Project VI). Explant assays will be used to determine the 'sponsiveness of DR axons to these guidance cues, and the ability of 5HT to modulate responsiveness. andidates identified in Project I DR transcriptome profiling will be examined in their functional assay. In Aim III, they will determine the influence of SERT genetic variants on the responseof TCAs and DR axons to the modulatory effects of 5HT on axon guidance molecule bioactivity. In collaboration with Project 3 (Blakely), they will determine how 5HT modulation of guidance cues is altered by functional SERT variants, and how SERT activity, independent of 5-HT, influences axon guidance. The role of SERT-interacting proteins ITGB3 and SYN1A as modulators of SERT modulation of axon guidance will be studied by in situ hybridization mapping, axon guidance assays using ITGB3 null mice, and disruption of SYN1A function.